Multiparameter flow cytometric analysis of CD45RA expression on Human peripheral blood leucocytes. Human whole blood was stained with BD Horizon™ BV421 Mouse Anti-Human CD45RO antibody (Cat. No. 562641/562649) and with either BD Horizon™ RB780 Mouse IgG2b, κ Isotype Control (Cat. No. 568741; Left Plots) or BD Horizon™ RB780 Mouse Anti-Human CD45RA antibody (Cat. No. 569081/569082; Right Plots). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). Bivariate pseudocolor density plots showing the correlated expression of either CD45RA (or Ig Isotype control staining) versus CD45RO (Top Plots) or side light-scatter (SSC-A) signals (Bottom Plots) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes (Top Plots) or leucocyte populations (Bottom Plots). Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
.png){kind=spacer}
Multiparameter flow cytometric analysis of CD45RA expression on Human peripheral blood leucocytes. Human whole blood was stained with BD Horizon™ BV421 Mouse Anti-Human CD45RO antibody (Cat. No. 562641/562649) and with either BD Horizon™ RB780 Mouse IgG2b, κ Isotype Control (Cat. No. 568741; Left Plots) or BD Horizon™ RB780 Mouse Anti-Human CD45RA antibody (Cat. No. 569081/569082; Right Plots). Erythrocytes were lysed with BD FACS™ Lysing Solution (Cat. No. 349202). Bivariate pseudocolor density plots showing the correlated expression of either CD45RA (or Ig Isotype control staining) versus CD45RO (Top Plots) or side light-scatter (SSC-A) signals (Bottom Plots) were derived from gated events with the forward and side light-scatter characteristics of intact lymphocytes (Top Plots) or leucocyte populations (Bottom Plots). Flow cytometry and data analysis were performed using a BD LSRFortessa™ X-20 Cell Analyzer System and FlowJo™ software.
Product Details
BD Horizon™
CD45R; PTPRC; LCA; Leukocyte common antigen
Human (QC Testing)
Mouse IgG2b, κ
Flow cytometry (Routinely Tested)
5 µl/test
IV N906
5788
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
Store undiluted at 4°C and protected from prolonged exposure to light. Do not freeze. The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. The antibody was conjugated to the dye under optimum conditions and unreacted dye was removed.
Recommended Assay Procedures
BD® CompBeads can be used as surrogates to assess fluorescence spillover (compensation). When fluorochrome conjugated antibodies are bound to BD® CompBeads, they have spectral properties very similar to cells. However, for some fluorochromes there can be small differences in spectral emissions compared to cells, resulting in spillover values that differ when compared to biological controls. It is strongly recommended that when using a reagent for the first time, users compare the spillover on cells and BD® CompBeads to ensure that BD® CompBeads are appropriate for your specific cellular application.
Product Notices
- When using high concentrations of antibody, background binding of this dye to erythroid fragments produced by ammonium chloride-based lysis, such as with BD Pharm Lyse™ Lysing Buffer (Cat. No. 555899), has been observed when the antibody conjugate was present during the lysis procedure. This may cause nonspecific staining of target cells, such as leukocytes, which have bound the resulting erythroid fragments. This background can be mitigated by any of the following: titrating the antibody conjugate to a lower concentration, fixing samples with formaldehyde, or removing erythrocytes before staining (eg, gradient centrifugation or pre-lysis with wash). This background has not been observed when cells were lysed with BD FACS™ Lysing Solution (Cat. No. 349202) after staining.
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- This reagent has been pre-diluted for use at the recommended Volume per Test. We typically use 1 × 10^6 cells in a 100-µl experimental sample (a test).
- An isotype control should be used at the same concentration as the antibody of interest.
- Please observe the following precautions: Absorption of visible light can significantly alter the energy transfer occurring in any tandem fluorochrome conjugate; therefore, we recommend that special precautions be taken (such as wrapping vials, tubes, or racks in aluminum foil) to prevent exposure of conjugated reagents, including cells stained with those reagents, to room illumination.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
- Human donor specific background has been observed in relation to the presence of anti-polyethylene glycol (PEG) antibodies, developed as a result of certain vaccines containing PEG, including some COVID-19 vaccines. We recommend use of BD Horizon Brilliant™ Stain Buffer in your experiments to help mitigate potential background. For more information visit https://www.bdbiosciences.com/en-us/support/product-notices.
- Please refer to http://regdocs.bd.com to access safety data sheets (SDS).
- Cy is a trademark of Global Life Sciences Solutions Germany GmbH or an affiliate doing business as Cytiva.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
RB780 Mouse IgG2b, κ Isotype Control RUO
Size
50 µg
Cat No.
568741
.png?imwidth=320)
BV421 Mouse Anti-Human CD45RO RUO
Size
100 Tests
Cat No.
562641
Lysing Solution 10X Concentrate IVD
Size
100 mL
Cat No.
349202
Lysing Buffer RUO
Size
100 mL
Cat No.
555899
569082 Rev. 3
Antibody Details
HI100
The HI100 monoclonal antibody specifically binds to the 220 kDa isoform of the human leukocyte common antigen, CD45RA. CD45RA is expressed on approximately 40-50% of peripheral CD4+ T cells, 50% of peripheral CD8+ T cells and on a portion of B cells and monocytes. The CD45RA antigen is expressed by naïve and activated T cells. CD45RA-specific antibodies are useful for the study of the suppressor/inducer subpopulation of CD4+ lymphocytes.
569082 Rev. 3
Format Details
RB780
The BD Horizon RealBlue™ 780 (RB780) Dye is part of the BD family of blue dyes. It is a tandem fluorochrome with an excitation maximum (Ex Max) at 498-nm and an emission maximum (Em Max) at 781-nm. Driven by BD innovation, RB780 can be used on both spectral and conventional cytometers and is designed to be excited by the Blue laser (488-nm) with minimal excitation by the 561-nm Yellow-Green laser. For conventional instruments equipped with a Blue laser (488-nm), RB780 can be used as an alternative to PE-Cy7 and we recommend using an optical filter centered near 780-nm (eg, a 780/60-nm bandpass filter). For spectral instruments equipped with a Blue laser (488-nm), it can be used in conjunction with PE-Cy7. RB780 is on average brighter than PE-Cy7 and has minimal spillover into Yellow-Green detectors.
RB780
Blue 488 nm
498 nm
781 nm
569082 Rev.3
Citations & References
View product citations for antibody "569082" on CiteAb
Development References (4)
-
Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:1-1182.
-
Koristka S, Cartellieri M, Theil A, et al. Retargeting of human regulatory T cells by single-chain bispecific antibodies.. J Immunol. 2012; 188(3):1551-8. (Clone-specific: Flow cytometry, Fluorescence activated cell sorting). View Reference
-
Schmidt RE. Non-lineage/natural killer section report: new and previously defined clusters. In: Knapp W. W. Knapp .. et al., ed. Leucocyte typing IV : white cell differentiation antigens. Oxford New York: Oxford University Press; 1989:517-542.
-
Zola H. Leukocyte and stromal cell molecules : the CD markers. Hoboken, N.J.: Wiley-Liss; 2007.
569082 Rev. 3
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.